Human natural killer (NK) cell activity is regulated by a family of killer-cell Ig-like receptors (KIR) for HLA class I. Combinations of KIR and HLA genotypes are associated with disease susceptibility, including sensitivity to virus infection and disorders of pregnancy. The goal of our study was to determine the extent to which peptides bound to HLA-C influence the specific binding of inhibitory KIR to group C1 or C2 HLA-C allotypes. Endogenous HLA-C*05:01-associated peptides were identified and tested for KIR binding to peptide-loaded HLA-C*05:01 (C2) and HLA-C*08:02 (C1), which are canonical ligands for KIR2DL1 and KIR2DL3, respectively, despite having only two amino acid differences. Specific binding of KIR2DL1 to the C2 allotype occurred with most peptides tested. In contrast, KIR2DL3 binding to the C1 allotype was restricted to a subset of peptides, providing an explanation for why KIR2DL3C1 interactions appear to be weaker than KIR2DL1C2. Furthermore, cross-reactive and functional binding of KIR2DL3 with the C2 allotype was even more restricted, occurring with only a few peptides. Moreover, we identified two peptides that, unexpectedly, promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. Our data show that KIR binding to HLA-C in the context of multiple peptides is a feature of strong specificity for HLA-C allotype C2, whereas greater selectivity for peptides is associated with more permissive reactivity with HLA-C allotypes. Peptide sequence-driven binding of KIR could lead to disease associations of KIR genotypes with specific HLA alleles, and be exploited by pathogens to modulate NK cell responses. The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We have reported that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C+ cells. Adding to the recent paradigm of signal transduction through higher-order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface. IL-15 is essential for NK cell survival, proliferation, and activation. Stimulation by IL-15 occurs through trans-presentation of IL-15, which is bound to the IL-15 receptor chain (IL-15R) expressed on other cells, to the IL-2 receptor and c chains expressed by NK cells. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, we tested if IL-15 transpresentation to human NK cells was sensitive to co-engagement of inhibitory receptors. We found that NK cell proliferation induced by IL-15 transpresentation was negatively regulated by co-engagement of either NKG2A or KIR inhibitory receptors. This negative regulation occurred selectively in the PI3KAktmTORC1S6 kinase pathway, as phosphorylation of Akt and ribosomal protein S6 were reduced upon co-engagement of inhibitory receptor, while phosphorylation of Stat5 and NK cell survival were not inhibited. These results reveal a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.